Abstract
Aims/hypothesis
IL-1β is a candidate mediator of apoptotic beta cell destruction, a process that leads to type 1 diabetes and progression of type 2 diabetes. IL-1β activates beta cell c-Jun N-terminal kinase (JNK), extracellular signal-regulated kinase (ERK) and p38, all of which are members of the mitogen-activated protein kinase (MAPK) family. Inhibition of JNK prevents IL-1β-mediated beta cell destruction. In mouse embryo fibroblasts and 3DO T cells, overexpression of the gene encoding growth arrest and DNA-damage-inducible 45β (Gadd45b) downregulates pro-apoptotic JNK signalling. The aim of this study was to investigate if Gadd45b prevents IL-1β-induced beta cell MAPK signalling and apoptosis.
Materials
Rat insulinoma INS-1E cells and mouse beta-TC3 cells stably expressing Gadd45b were generated. The effects of Gadd45b expression on signalling by JNK, ERK and p38 were assessed by Western blotting and kinase assays. Apoptosis rate was measured by terminal deoxynucleotidyl-mediated dUTP-biotin nick end-labelling (TUNEL) and an ELISA designed to detect apoptotic nucleosomes. Expression of endogenous Gadd45b mRNA was measured by RT-PCR.
Results
In INS-1E and beta-TC3 cells, expression of Gadd45b inhibited IL-1β-induced activation of JNK and ERK, but augmented IL-1β-mediated p38 activity. IL-1β-induced nitric oxide production and decreases in insulin content and secretion were reduced by GADD45β. IL-1β-induced apoptosis was reduced by GADD45β by up to 77%. Although IL-1β stimulated the time-dependent induction of endogenous Gadd45b in INS-1E cells and rat islets, expression levels were lower in these cells than in IL-1β-exposed NIH-3T3 and 3DO T cells.
Conclusions/interpretation
Inadequate induction of Gadd45b, which encodes a novel beta cell JNK and ERK inhibitor, may in part explain the pro-apoptotic response of beta cells to IL-1β.
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Introduction
Destruction of the pancreatic beta cells is the hallmark of type 1 diabetes [1] and is involved in the development and progression of type 2 diabetes [2]. The cytokine IL-1β has been proposed to be a mediator of the autoimmune destruction that leads to type 1 diabetes [3] and it has recently been suggested that IL-1β is involved in the glucotoxic beta cell destruction associated with type 2 diabetes [4].
In beta cells IL-1β causes activation of the c-Jun N-terminal kinase (JNK), extracellular signal-regulated kinase (ERK) and p38, members of the mitogen-activated protein kinase (MAPK) family of threonine or serine kinases [5, 6]. ERK and p38 are both involved in the IL-1β-mediated beta cell induction of inducible nitric oxide (NO) synthase (iNOS), leading to NO formation [6]. NO is a secondary messenger for the beta cell cytotoxic effect of IL-1β [7, 8], especially in rodent islets. However, NO-independent cytokine-mediated destruction of beta cells is possible [9–11] and of particular relevance in human beta cells [9, 11]. Inhibition of JNK activation protects beta cell lines against IL-1β-induced apoptosis [5, 12] and human islets against the destruction mediated by IL-1β, TNF-α and IFN-γ [13, 14]. Disruption of JNK activation also protects beta cells against T-cell-mediated killing [15], increases islet survival after isolation [13, 16] and improves islet graft function [16, 17]. Taken together, these observations demonstrate that JNK inhibition is crucial in maintaining the function and survival of beta cells when exposed to multiple stressors.
Prolonged JNK activation promotes apoptosis in several cell types [18, 19]. IL-1β alone or in combination with TNF-α and IFN-γ causes sustained activation of JNK in beta cells [13, 20], indicating insufficient downregulation of JNK activity in cytokine-exposed beta cells.
Gadd45b (which encodes growth arrest and DNA-damage-inducible 45β), Gadd45a and Gadd45g are members of the Gadd45 gene family, which is involved in growth arrest, apoptosis and DNA repair [21]. We have recently shown that overexpression of Gadd45b downregulates pro-apoptotic JNK signalling in mouse embryo fibroblasts (MEFs) and 3DO T cells, without affecting the ERK and p38 activities [22]. However, the involvement of Gadd45b in MAPK signalling seems to be cell-specific, because overexpression of Gadd45b in other cell types augments JNK and p38 activities and promotes apoptosis [23].
The aim of the present work was therefore to investigate the regulation of Gadd45b induction by IL-1β in beta cells and to explore the involvement of GADD45β in beta cell IL-1β-induced MAPK signalling and apoptosis. We report that Gadd45b is a novel beta cell primary response gene with insufficient induction in INS-1E and beta-TC3 cell IL-1β signalling, and that expression of Gadd45b inhibits IL-1β-induced JNK and ERK signalling and apoptosis.
Materials and methods
Reagents
Recombinant mouse IL-1β was from BD Pharmingen (Erembodegen, Belgium), Recombinant human TNF-α was from Endogen (Woburn, MA, USA) and recombinant rat IFN-γ from R & D Systems (Minneapolis, MN, USA). Phorbol myristate acetate (PMA) and cycloheximide (CHX) were from Sigma-Aldrich (San Diego, CA, USA).
Cell culture
INS-1E cells (kindly provided by C. Wollheim, University Medical Centre, Geneva, Switzerland), beta-TC3 cells and 3DO T cells were grown in RPMI-1640 medium (11 mmol/l glucose) containing 10% FCS, 100 U/ml penicillin and 100 μg/ml streptomycin. In addition, the INS-1E and 3DO T-cell culture medium contained 50 μmol/l β-mercaptoethanol. Mouse NIH-3T3 fibroblasts were maintained in DMEM supplied with FCS, penicillin and streptomycin as described above.
Islet isolation and culture
Pancreatic islets of Langerhans from 3- to 5-day-old Wistar Furth rats (Charles River, Sulzfeldt, Germany) were isolated by hand-picking after collagenase digestion as described previously [6]. Islets were cultured in RPMI-1640 supplemented with 10% FCS, 100 U/ml penicillin, 100 μg/ml streptomycin and 0.038% NaHCO3 for 7 days prior to experimentation.
Stable transfection
INS-1E and beta-TC3 cells were transfected with pcDNA3.1/Flag (Invitrogen, Carlsbad, CA, USA) or pcDNA3.1/Flag-Gadd45b [24] by the use of FuGENE 6 Transfection Reagent (Roche, Basel, Switzerland) following the manufacturer’s instructions. The cells were cultured for 2 days, then trypsinised and reseeded in 100 mm dishes at two densities (1:10 and 9:10). Based on kill-curve experiments, 100 μg/ml G418 (Life technologies, Grand Island, NY, USA) was added for the selection of geniticin-resistant cells. Geniticin-resistant colonies were selected after 2–4 weeks and propagated in 50 μg/ml G418. The selected clones were assayed for Gadd45b (Fig. 1) expression by RT-PCR and Western blotting.
Protein extraction
Following IL-1β stimulation 5×105 INS-1E or beta-TC3 cells were washed once in cold PBS and then lysed in 75 μl lysis buffer containing 20 mmol/l Tris acetate, pH 7.0, 0.27 mol/l sucrose, 1 mmol/l EDTA, 1 mmol/l EGTA, 1 mmol/l Na3VO4, 50 mmol/l NaF, 1% Triton X-100, 5 mmol/l sodium pyrophosphate, 10 mmol/l β-glycerophosphate, 1 mmol/l dithiothreitol, 1 mmol/l benzamidine and 4 μg/ml leupeptin. The detergent-insoluble material was pelleted by centrifugation at 15,000× g for 5 min at 4°C. The protein concentration in the supernatant was measured by Bradford assay (Bio-Rad, Hercules, CA, USA). The supernatants containing whole-cell lysate were either used immediately (for kinase assay or Western blotting) or stored at −80°C.
Western blotting
Protein (15–20 μg) was separated on 10% BisTris gels (Invitrogen, Carlsbad, USA) by gel electrophoresis using NuPAGE technology (Invitrogen). Following electrotransfer to nitrocellulose membranes (Invitrogen) and blocking in 5% milk dissolved in Tris-buffered saline containing 0.1% Tween-20, membranes were incubated in primary antibody at 4°C overnight. The following antibodies were used: rabbit anti-JNK (diluted 1:1000; Cell Signaling Technology, Beverly, MA, USA); rabbit anti-phospho-JNK (1:1000; Cell Signaling Technology); rabbit anti-ERK (1:1000; Cell Signaling Technology); rabbit anti-phospho-ERK (1:1000; Cell Signaling Technology); rabbit anti-p38 (1:500; Cell Signaling Technology); rabbit anti-phospho-p38 (1:500; Cell Signaling Technology); rabbit anti-phospho-MKK7 (1:250; Cell Signaling Technology); mouse anti-IκBα (1:500; Active Motif, Rixensart, Belgium); mouse anti-iNOS (1:5000; BD Biosciences, San Jose, CA, USA); mouse anti-actin (1:10 000; Abcam, Cambridge, UK) and rabbit anti-GADD45α (1:500; Santa Cruz Biotechnology, Santa Cruz, CA, USA). Mouse anti-GADD45β (1:500) was used as described previously [22]. Following addition of secondary peroxidase-conjugated anti-rabbit or -mouse antibodies (both from Cell Signaling Technology), binding of antibody was detected with enhanced chemiluminescence using SuperSignal (Pierce, Rockford, IL, USA). Bands were visualised with a Luminescent Image Analyzer LAS-3000 and the optical densities were quantitated using Multi Gauge Software (both from Fujifilm, Stamford, CT, USA).
Kinase assay
The kinase assays measuring phosphotransferase activities in the cell lysates towards exogenous c-Jun (Calbiochem, San Diego, CA, USA), Elk-1 (a kind gift from K. Seedorf, Lilly, Hamburg) and Hsp25 (Stressgen, Victoria, BC, Canada) were performed as described previously [6] except that 2 μg GST-c-Jun was used instead of activating transcription factor-2. The incorporation of γ-32P from [γ-32P]ATP (Amersham, UK) in the substrates was measured by autoradiography and quantitated using Multi Gauge Software (Fujifilm Medical systems).
RNA extraction and cDNA synthesis
Total RNA from 1×106 INS-1E and beta-TC3 cells, 5×105 NIH-3T3 and 3DO T cells or 300 islets was extracted by the use of Trizol reagent (Invitrogen) following the manufacturer’s instructions. cDNA was synthesised from 400 ng of RNA using the TaqMan kit from Perkin Elmer (Wellesley, MA, USA).
Quantitative RT-PCR
Real time RT-PCR was performed on the 7900 HT Sequence Detection System (Applied Biosystems, Foster City, CA, USA) using cDNA and SYBR Green master mix (Applied Biosystems). Expression levels of Gadd45a, Gadd45b and Gadd45g were calculated by the standard curve method and the quantity of each cDNA was normalised for transcription factor Sp-1, showing no regulation by IL-1β [25]. The primers were as follows: Gadd45a, 5′-TGAGCTGCTGCTACTGGAGA-3′ and 5′-TGTGATGAATGTGGGTTCGT-3′; Gadd45b, 5′-ATTGACATCGTCCGGGTATC-3′ and 5′-TGACAGTTCGTGACCAGGAG-3′; Gadd45g, 5′-GCATCCTCATTTCGAATCCT-3′ and 5’-CACCCAGTCGTTGAAGCTG-3′; Sp-1, 5′-GGCTACCCCTACCTCAAAGG-3′ and 5′-CACAACATACTGCCCACCAG-3′.
NO synthesis
INS-1E NO production was measured as nitrite accumulation in conditioned medium determined by the Griess reaction, as described previously [6]. The detection limit was 1 μmol/l.
Insulin content and secretion
The insulin content of protein extracts from INS-1E and beta-TC3 cells and the accumulated insulin secretion, measured as insulin in the conditioned medium, were determined by radioimmunoassay [6].
Detection of apoptosis
To determine the apoptosis rate two assays were used.
For the Cell Death Detection ELISAplus kit (Roche), which detects apoptotic nucleosomes in the cytoplasmic fraction of cell lysate, 5×104 INS-1E cells were seeded in 48-well dishes. Following exposure to IL-1β, the assay was performed as described by the manufacturer. The nucleosomes were measured by sandwich ELISA and data are presented as fold induction relative to untreated cells. For the detection of apoptosis with the TUNEL (terminal deoxynucleotidyl-mediated dUTP-biotin nick end-labelling) assay, 2×105 INS-1E cells were seeded in two-chamber wells. Following exposure to IL-1β, cells were fixed in 4% paraformaldehyde and the free 3′-OH strand breaks were detected by the TUNEL labelling technique according to the manufacturer’s instructions (ApopTag In Situ Apoptosis Detection Kit; Chemicon International, Temecula, CA, USA). Staining with DAPI (4′,6-diamidino-2-phenylindole; 1 μg/ml) was used to assess the total number of cells. By fluorescence microscopy, a total of at least 500 cells in each condition were counted in a randomised manner by an investigator (M.G.D.) blinded to the treatment conditions. The number of TUNEL-positive cells was expressed as the percentage of the number of DAPI-stained cells.
Statistical analysis
In histograms, data are mean±SEM. Statistical analysis was performed with the two-tailed paired Student’s t test and p<0.05 was chosen as the level of significance.
Results
Selection of Gadd45b-expressing INS-1E clones
To investigate the level of GADD45β production in the stably transfected INS-1E clones, we performed Western blotting of five clones. As seen in Fig. 1a, clones 1 and 5 produced the highest levels of the GADD45β protein, with similar findings at the mRNA level (Fig. 1b). To investigate a possible dose-dependency of the effects of Gadd45b expression, clones 1 and 2 were chosen for further experiments.
GADD45β attenuates IL-1β-induced JNK and ERK activation, but augments p38 activation
To determine the involvement of GADD45β in the regulation of IL-1β-induced MAPK signalling, we first performed Western blotting with phosphospecific antibodies recognising the phosphorylated (activated) kinases. As shown in Fig. 2a,b, high levels of GADD45β production (clone 1) reduced IL-1β-induced INS-1E JNK activation by 60%. GADD45β production dose-dependently inhibited IL-1β-induced ERK activity by up to 86%. On the other hand, high levels of GADD45β production augmented both basal and IL-1β-mediated p38 MAPK activation, by 400 and 70%, respectively. Similar inhibition of IL-1β-induced JNK and ERK activation, but less enhancement of p38 activation, was detected in a pool of GADD45β-producing mouse insulinoma beta-TC3 cells (Fig. 2c,d).
To examine the in vitro kinase activities of JNK, ERK and p38 MAPK in Gadd45b-expressing INS-1E cells, we determined the phosphotransferase activities toward exogenous c-Jun, Elk-1 and Hsp25 [6]. GADD45β dose-dependently reduced IL-1β-induced, ERK-mediated Elk-1 phosphorylation by up to 70% (Fig. 3). IL-1β-induced c-Jun phosphorylation mediated by JNK was inhibited by 54% by high levels of GADD45β (clone 1). IL-1β-mediated p38 MAPK activity, measured by the phosphorylation of p38-activated MAPK-activated protein kinase 2 substrate Hsp25, was enhanced by up to 47% by Gadd45b expression.
GADD45β decreases IL-1β-induced MKK7 activation but not inhibitor protein κBα degradation
We have previously shown that GADD45β inhibits JNK signalling in 3DO T cells and MEFs via inhibition of the JNK upstream kinase MAPK kinase (MKK) 7 [24]. Similarly, high levels of GADD45β production decreased the IL-1β-induced phosphorylation of MKK7 in INS-1E cells (Fig. 4). Having found that GADD45β modulated IL-1β-induced INS-1E MAPK signalling, we now asked whether GADD45β could interact with nuclear factor κB (NFκB) signalling, another major beta cell IL-1β signalling pathway [26]. As shown in Fig. 4, GADD45β production did not affect IL-1β-mediated degradation of the NFκB repressor inhibitor protein κBα (IκBα).
GADD45β production decreases IL-1β-induced NO synthesis and inhibition of insulin content and release
To assess the effect of stable Gadd45b expression on INS-1E cell function, we first measured NO accumulation in the medium following 48 h of IL-1β exposure. As shown in Fig 5b, low and high concentrations of GADD45β decreased IL-1β-induced NO accumulation by 50% and 72%, respectively. The reduced amount of IL-1β-induced NO accumulation by Gadd45b-expressing cells was due to decreased production of iNOS protein (Fig. 5a).
Forty-eight hours of exposure to IL-1β caused 82% and 72% reduction of INS-1E cell insulin content (Fig. 5c) and accumulated insulin release (Fig. 5d), respectively. Stable Gadd45b expression neither affected the insulin content (Fig. 5c) nor the accumulated insulin release (Fig. 5d) of control cell cultures; however, high levels of GADD45β production reduced the IL-1β-mediated inhibition of insulin content and release to 47% and 38%, respectively, (Fig. 5c,d). In beta-TC3 cells, 48 h of IL-1β exposure caused a 57% reduction in both insulin content (Fig. 5e) and accumulated insulin release (Fig. 5f). The insulin content and accumulated insulin release were unaffected in the control pool culture of Gadd45b-expressing beta-TC3 cells, but the IL-1β-induced inhibition of insulin content and release was reduced to 27 and 23%, respectively, (Fig. 5e,f).
GADD45β production reduces IL-1β-induced INS-1E apoptosis
Forty-eight hours of incubation with IL-1β caused 4.7- and 4.9-fold increases in the apoptosis rate in mock transfected INS-1E cells investigated with the Cell Death Detection ELISAplus and TUNEL methods, respectively, (Fig. 6a,b). High levels of GADD45β production reduced the apoptosis rate by 77% and 60%, assayed by Cell Death Detection ELISAplus and TUNEL assays, respectively, (Fig. 6a,b). In the TUNEL assay, the basal apoptosis rate was not affected by GADD45β production.
Induction of Gadd45 gene family by cytokines in islets and INS-1E cells
Having investigated the involvement of Gadd45b in MAPK signalling, we now explored the regulation of endogenous Gadd45b by cytokines. As seen in Fig. 7a, IL-1β caused time-dependent induction of Gadd45b mRNA in INS-1E cells. Induction occurred within 1 h, there was a 10.9-fold peak of induction at 2 h, and the basal level was regained at 24 h. TNF-α did not induce Gadd45bmRNA and IFN-γ caused a weaker and more delayed induction of Gadd45b, with maximal 4.7-fold induction of Gadd45b mRNA at 4 h. Unlike Gadd45b, the Gadd45a and Gadd45g gene family members were not inducible by IL-1β, TNF-α or IFN-γ in INS-1E cells. The time-dependent induction of Gadd45b mRNA by IL-1β was reproduced in isolated rat islets. Induction was 3.7-fold at 30 min and maximal (29.8-fold) at 2 h, and sustained induction was detectable until at least 24 h of IL-1β exposure (Fig. 7b). The changes at mRNA level were mirrored at the protein level, as the GADD45α protein showed no regulation by IL-1β in INS-1E cells and islets (Fig. 7c), and the GADD45β protein was upregulated by IL-1β in both islets and INS-1E cells in a time-dependent manner (Fig. 7c). However, maximal production of endogenous GADD45β protein did not exceed the amount produced by clones 1 and 2 (Fig. 7c,d). The anti-GADD45γ antibody was not suitable for Western blotting.
Gadd45b, but not Gadd45a or Gadd45g, is a primary response gene in beta cell IL-1β signalling
To further investigate the regulation of Gadd45b mRNA expression, INS-1E cells were preincubated with the protein synthesis inhibitor CHX and then exposed to IL-1β or the phorbol ester PMA [27]. All three Gadd45b genes were induced by CHX, with 5.5-, 13.6- and 12.3-fold induction of Gadd45a, Gadd45b and Gadd45g, respectively, (Fig. 8). As expected, Gadd45b, but not Gadd45a or Gadd45g, was induced by IL-1β. PMA caused induction of Gadd45b mRNA but not of Gadd45a or Gadd45g mRNAs. Coincubation of CHX and IL-1β caused 4.4-fold superinduction of Gadd45b over the IL-1β-treated level. No superinduction of Gadd45a or Gadd45g was seen on coincubation of CHX and IL-1β. None of the Gadd45 genes showed superinduction on coincubation with CHX and PMA. Induction of Gadd45b mRNA by IL-1β, but not by PMA, is thus refractory to translational blockade and Gadd45b is a primary response gene in IL-1β, but not PMA, signalling [28]. Neither Gadd45a nor Gadd45g is a primary response gene in beta cell IL-1β or PMA signalling.
Reduced Gadd45b induction in beta-TC3 and INS-1E cells compared with NIH-3T3 and 3DO T cells
To compare the induction of Gadd45b mRNA in different species and cell types, mouse beta-TC3, rat INS-1E, mouse fibroblast NIH-3T3 and mouse 3DO T cells were exposed to IL-1β or PMA. As seen in Fig. 9a, IL-1β caused time-dependent induction of Gadd45b mRNA in all four cell lines, with maximal induction of 51.0-, 39.5-, 11.8-, 4.4-fold in 3DO, NIH-3T3, INS-1E and beta-T3 cells, respectively. PMA was a weaker inducer of Gadd45b mRNA and gave greater similarity of induction of Gadd45b mRNA in the four cell lines, with maximal induction of 13.1-, 9.2-, 7.1- and 5.4-fold in the 3DO, NIH-3T3, INS-1E and beta-T3 cells, respectively. This indicates insufficient Gadd45b induction specifically in response to IL-1β in beta cells.
The high levels of IL-1β-induced Gadd45b expression found in 3DO T cells were associated with transient IL-1β-mediated JNK activation, which was terminated within 1 h (Fig. 9b), whereas the lower level of IL-1β-induced Gadd45b expression in INS-1E cells was associated with sustained JNK activation lasting for at least 8 h (Fig. 9c).
Discussion
The molecular mechanisms for the noticeable sensitivity of beta cells to apoptosis induced by IL-1β and the potentiating cytokines TNF-α and IFN-γ are incompletely understood, but include IL-1β-mediated downregulation of islet-brain 1 protein [29] and inadequate upregulation of manganese superoxide dismutase [30]. In the present paper we report decreased upregulation of beta cell Gadd45b by IL-1β, and that stable expression of Gadd45b inhibits IL-1β-induced MAPK activation, NO synthesis and apoptosis, and restores the IL-1β-induced decreases in insulin content and release.
Whereas GADD45α and GADD45γ are strictly pro-apoptotic proteins [23, 31], the involvement of GADD45β in the apoptotic process is debated. The present study demonstrates an anti-apoptotic effect of Gadd45b expression in beta cells exposed to IL-1β, and we have previously shown that enhanced Gadd45b expression prevents apoptosis [22, 24, 32] in other cell types. However, others have shown a pro-apoptotic effect of Gadd45b expression [23], whereas in two recent studies Gadd45b deficiency [33] or overexpression [31] had no effect on cell survival or apoptosis. The molecular switch that determines the anti- or pro-apoptotic effects of GADD45β seems to be the ability of GADD45β in a particular cell type to either inhibit or activate JNK signalling. The pro-apoptotic action of GADD45β depends on the activation of JNK by MKK7-independent signalling via activation of MAPK kinase kinase (MEKK) 4, leading to MKK4 phosphorylation and concomitant JNK activation [23]. The GADD45β-mediated inhibition of JNK activity (Figs. 2 and 3) is probably linked to the observed decrease in MKK7 activation, a known target of GADD45β inhibition (Fig. 4) [24]. Inhibition of JNK has consistently been associated with decreased beta cell apoptosis [5, 12–14, 29] and is thus the probable major mechanism behind the observed beta-cell-protective properties of GADD45β. The decreased apoptosis rate observed in Gadd45b-expressing INS-1E cells is not likely to be a consequence of the decreased NO production, as IL-1β-induced destruction of INS-1E cells [10] is independent of NO, as in human islets [9, 11].
The GADD45β-mediated inhibition of apoptosis in IL-1β-exposed INS-1E cells was not complete (60–77%; Fig. 6a,b). This is probably due to the incomplete JNK inhibition caused by the residual MKK7 activity found even at high levels of Gadd45b expression or to JNK activation mediated by MKK4 (Fig. 4). If higher levels of Gadd45b expression had been achieved, we would have expected a higher degree of MKK7 and JNK inhibition with concomitant inhibition of INS-1E apoptosis. However, other beta cell pro-apoptotic signalling pathways not affected by Gadd45b expression, such as the NFκB pathway (Fig. 4) [26, 34], may still signal apoptosis in the context of enhanced Gadd45b expression.
The decrease in iNOS and NO synthesis by Gadd45b expression (Fig. 5a,b) may be mediated via ERK, as ERK inhibition decreased IL-1β-induced rat islet iNOS and NO production [6], whereas IL-1β-induced iNOS expression was independent of JNK inhibition [35]. The augmentation of p38 activation by GADD45β in INS-1E cells (Figs. 2 and 3) may explain why IL-1β-induced NO production was decreased by only 75%, as p38 activation is required for beta cell NO production [6]. The mechanism behind the observed p38 activation seems to be via GADD45β-mediated MEKK4 activation, as shown by others [23, 36, 37]. Inhibition of ERK activity by GADD45β in INS-1E cells is a novel observation, as previous studies have shown no effect on ERK activity by GADD45β [22, 23]. The mechanism of action is unclear, as IL-1β-induced beta cell ERK activation signals via MAPK/ERK kinase (MEK) 1/2 and not MKK7, on the basis of studies with the MEK inhibitor [6]. GADD45β inhibition of the ERK upstream kinases ras and raf is a possible mechanism, but this has not been investigated.
The GADD45β-mediated preservation of insulin content and release following IL-1β exposure (Fig. 5c–f) may be a consequence of the decreased apoptosis rate in these cells; however, JNK activation has been reported to suppress insulin gene expression and secretion in rat islets independently of cell death [17]. ERK does not seem to be involved in glucose-induced insulin release [38].
Expression of Gadd45b is regulated by the NFκB signalling pathway [22, 27, 31]. The superinduction of Gadd45b mRNA mediated by co-exposure of IL-1β and CHX (Fig. 8) suggests that Gadd45b is also regulated by NFκB in INS-1E cells. Whereas NFκB in general signals cell survival [39], NFκB signals apoptosis in beta cells [26, 34].
A limitation of this study is that it was performed in a rodent beta cell line; however, in human islets JNK and ERK also signal beta cell destruction [13, 14, 40], whereas p38 signals beta cell survival [14], making GADD45β a potentially strong tool for the protection of human beta cells against cytokine-mediated destruction. Testing this hypothesis is limited by the efficiency of transfecting human islets and the lack of small molecules able to modulate Gadd45b expression, but will be a future area of research.
We conclude that Gadd45b is a novel primary response gene in beta cell IL-1β signalling, and our finding that GADD45β is a beta cell inhibitor of JNK and ERK that is insufficiently induced may be part of the explanation for the beta cell pro-apoptotic response to IL-1β.
Abbreviations
- CHX:
-
cycloheximide
- ERK:
-
extracellular signal-regulated kinase
- GADD:
-
growth arrest and DNA-damage-inducible
- iNOS:
-
inducible nitric oxide synthase
- IκBα:
-
inhibitor protein κBα
- JNK:
-
c-Jun N-terminal kinase
- MAPK:
-
mitogen-activated protein kinase
- MEF:
-
mouse embryo fibroblast
- MEK:
-
mitogen-activated protein kinase/extracellular signal-regulated kinase
- MKK:
-
mitogen-activated protein kinase kinase
- MEKK:
-
mitogen-activated protein kinase kinase kinase
- NFκB:
-
nuclear factor κB
- NO:
-
nitric oxide
- PMA:
-
phorbol myristate acetate
References
Atkinson MA, Maclaren NK (1994) The pathogenesis of insulin-dependent diabetes mellitus. N Engl J Med 331:1428–1436
Butler AE, Janson J, Bonner-Weir S, Ritzel R, Rizza RA, Butler PC (2003) Beta-cell deficit and increased beta-cell apoptosis in humans with type 2 diabetes. Diabetes 52:102–110
Eizirik DL, Mandrup-Poulsen T (2001) A choice of death—the signal-transduction of immune-mediated beta-cell apoptosis. Diabetologia 44:2115–2133
Maedler K, Sergeev P, Ris F et al (2002) Glucose-induced beta cell production of IL-1beta contributes to glucotoxicity in human pancreatic islets. J Clin Invest 110:851–860
Ammendrup A, Maillard A, Nielsen K et al (2000) The c-Jun amino-terminal kinase pathway is preferentially activated by interleukin-1 and controls apoptosis in differentiating pancreatic beta-cells. Diabetes 49:1468–1476
Larsen CM, Wadt KA, Juhl LF et al (1998) Interleukin-1 beta-induced rat pancreatic islet nitric oxide synthesis requires both the p38 and extracellular signal-regulated kinase 1/2 mitogen-activated protein kinases. J Biol Chem 273:15294–15300
Corbett JA, McDaniel ML (1994) Reversibility of interleukin-1 beta-induced islet destruction and dysfunction by the inhibition of nitric oxide synthase. Biochem J 299:719–724
Thomas HE, Darwiche R, Corbett JA, Kay TW (2002) Interleukin-1 plus gamma-interferon-induced pancreatic beta-cell dysfunction is mediated by beta-cell nitric oxide production. Diabetes 51:311–316
Delaney CA, Pavlovic D, Hoorens A, Pipeleers DG, Eizirik DL (1997) Cytokines induce deoxyribonucleic acid strand breaks and apoptosis in human pancreatic islet cells. Endocrinology 138:2610–2614
Kutlu B, Cardozo AK, Darville MI et al (2003) Discovery of gene networks regulating cytokine-induced dysfunction and apoptosis in insulin-producing INS-1 cells. Diabetes 52:2701–2719
Loweth AC, Williams GT, James RF, Scarpello JH, Morgan NG (1998) Human islets of Langerhans express Fas ligand and undergo apoptosis in response to interleukin-1beta and Fas ligation. Diabetes 47:727–732
Bonny C, Oberson A, Negri S, Sauser C, Schorderet DF (2001) Cell-permeable peptide inhibitors of JNK: novel blockers of beta-cell death. Diabetes 50:77–82
Aikin R, Maysinger D, Rosenberg L (2004) Cross-talk between PI3K/AKT and JNK mediates survival of isolated human islets. Endocrinology 145:4522–4531
Eckhoff DE, Smyth CA, Eckstein C et al (2003) Suppression of the c-Jun N-terminal kinase pathway by 17beta-estradiol can preserve human islet functional mass from proinflammatory cytokine-induced destruction. Surgery 134:169–179
Jaeschke A, Rincon M, Doran B et al (2005) Disruption of the Jnk2 (Mapk9) gene reduces destructive insulitis and diabetes in a mouse model of type I diabetes. Proc Natl Acad Sci USA 102:6931–6935
Noguchi H, Nakai Y, Matsumoto S et al (2005) Cell permeable peptide of JNK inhibitor prevents islet apoptosis immediately after isolation and improves islet graft function. Am J Transplant 5:1848–1855
Kaneto H, Xu G, Fujii N, Kim S, Bonner-Weir S, Weir GC (2002) Involvement of c-Jun N-terminal kinase in oxidative stress-mediated suppression of insulin gene expression. J Biol Chem 277:30010–30018
Tobiume K, Matsuzawa A, Takahashi T et al (2001) ASK1 is required for sustained activations of JNK/p38 MAP kinases and apoptosis. EMBO Rep 2:222–228
Xia Z, Dickens M, Raingeaud J, Davis RJ, Greenberg ME (1995) Opposing effects of ERK and JNK-p38 MAP kinases on apoptosis. Science 270:1326–1331
Storling J, Zaitsev SV, Kapelioukh IL et al (2005) Calcium has a permissive role in interleukin-1beta-induced c-Jun N-terminal kinase activation in insulin-secreting cells. Endocrinology 146:3026–3036
Liebermann DA, Hoffman B (2003) Myeloid differentiation (MyD) primary response genes in hematopoiesis. Blood Cells Mol Dis 31:213–228
De Smaele E, Zazzeroni F, Papa S et al (2001) Induction of gadd45beta by NF-kappaB downregulates pro-apoptotic JNK signalling. Nature 414:308–313
Takekawa M, Saito H (1998) A family of stress-inducible GADD45-like proteins mediate activation of the stress-responsive MTK1/MEKK4 MAPKKK. Cell 95:521–530
Papa S, Zazzeroni F, Bubici C et al (2004) Gadd45beta mediates the NF-kappaB suppression of JNK signalling by targeting MKK7/JNKK2. Nature Cell Biol 6:146–153
Wadt KA, Larsen CM, Andersen HU, Nielsen K, Karlsen AE, Mandrup-Poulsen T (1998) Ciliary neurotrophic factor potentiates the beta-cell inhibitory effect of IL-1beta in rat pancreatic islets associated with increased nitric oxide synthesis and increased expression of inducible nitric oxide synthase. Diabetes 47:1602–1608
Heimberg H, Heremans Y, Jobin C et al (2001) Inhibition of cytokine-induced NF-kappaB activation by adenovirus-mediated expression of a NF-kappaB super-repressor prevents beta-cell apoptosis. Diabetes 50:2219–2224
Jin R, De Smaele E, Zazzeroni F et al (2002) Regulation of the gadd45beta promoter by NF-kappaB. DNA Cell Biol 21:491–503
Herschman HR (1991) Primary response genes induced by growth factors and tumor promoters. Annu Rev Biochem 60:281–319
Bonny C, Oberson A, Steinmann M, Schorderet DF, Nicod P, Waeber G (2000) IB1 reduces cytokine-induced apoptosis of insulin-secreting cells. J Biol Chem 275:16466–16472
Hohmeier HE, Thigpen A, Tran VV, Davis R, Newgard CB (1998) Stable expression of manganese superoxide dismutase (MnSOD) in insulinoma cells prevents IL-1beta-induced cytotoxicity and reduces nitric oxide production. J Clin Invest 101:1811–1820
Zerbini LF, Wang Y, Czibere A et al (2004) NF-kappa B-mediated repression of growth arrest- and DNA-damage-inducible proteins 45alpha and gamma is essential for cancer cell survival. Proc Natl Acad Sci USA 101:13618–13623
Zazzeroni F, Papa S, Algeciras-Schimnich A et al (2003) Gadd45 beta mediates the protective effects of CD40 costimulation against Fas-induced apoptosis. Blood 102:3270–3279
Amanullah A, Azam N, Balliet A et al (2003) Cell signalling: cell survival and a Gadd45-factor deficiency. Nature 424:741
Giannoukakis N, Rudert WA, Trucco M, Robbins PD (2000) Protection of human islets from the effects of interleukin-1beta by adenoviral gene transfer of an Ikappa B repressor. J Biol Chem 275:36509–36513
Nikulina MA, Sandhu N, Shamim Z et al (2003) The JNK binding domain of islet-brain 1 inhibits IL-1 induced JNK activity and apoptosis but not the transcription of key proapoptotic or protective genes in insulin-secreting cell lines. Cytokine 24:13–24
Chi H (2004) GADD45beta/GADD45gamma and MEKK4 comprise a genetic pathway mediating STAT4-independent IFNgamma production in T cells. EMBO J 23:1576–1586
Lu B, Ferrandino AF, Flavell RA (2004) Gadd45beta is important for perpetuating cognate and inflammatory signals in T cells. Nature Immunol 5:38–44
Khoo S, Cobb MH (1997) Activation of mitogen-activating protein kinase by glucose is not required for insulin secretion. Proc Natl Acad Sci USA 94:5599–5604
Barkett M (1999) Control of apoptosis by Rel/NF-kappaB transcription factors. Oncogene 18:6910–6924
Maedler K, Størling J, Sturis J et al (2004) Glucose- and interleukin-1beta-induced beta-cell apoptosis requires Ca2+ influx and extracellular signal-regulated kinase (ERK) 1/2 activation and is prevented by a sulfonylurea receptor 1/inwardly rectifying K+ channel 6.2 (SUR/Kir6.2) selective potassium channel opener in human islets. Diabetes 53:1706–1713
Acknowledgements
We would like to thank A.-S. Hillesø and H. Foght for excellent technical assistance. This work was supported by the Danish Diabetes Association and the JDRF Center for Prevention of Beta Cell Destruction in Europe under grant 4-2002-457.
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Larsen, C.M., Døssing, M.G., Papa, S. et al. Growth arrest- and DNA-damage-inducible 45β gene inhibits c-Jun N-terminal kinase and extracellular signal-regulated kinase and decreases IL-1β-induced apoptosis in insulin-producing INS-1E cells. Diabetologia 49, 980–989 (2006). https://doi.org/10.1007/s00125-006-0164-0
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DOI: https://doi.org/10.1007/s00125-006-0164-0